Natural compound-mediated activation of inducible Nrf2 enhances the cellular antioxidant levels and suppresses ROS, which can be used as a strategy to inhibit Cr(VI)-induced malignant transformation, representing the first stage of Cr(VI) carcinogenesis. Our preliminary studies from the parent R01 grant show that after transformation these cells exhibit constitutive Nrf2 activation, which up-regulates its target proteins, including SOD and Bcl-2, leading to cell survival and tumorigenesis, signifying the second stage of metal or Cr(VI) carcinogenesis. Thus, our data indicate that Nrf2 can be an important target for prevention against Cr(VI) carcinogenesis at both stages. The parent R01 proposal investigates the protection of a natural compound against Cr(VI)-induced cell transformation, Cr(VI)-transformed cells-induced tumorigenesis, and Cr(VI)-induced angiogenesis and tumor formation with overall theme focused on endpoints of the protection aganist Cr(VI)-induced carcinogenesis. This ViCTER application expands the scope of the parent R01 through developing new transdisciplinary collaboration by engaging with experts in the areas of autophagy, metal carcinogenesis, and mechanism-based cancer prevention to focus on mechanistic aspect on Cr(VI)-induced carcinogenesis and its protection using a natural compound. This study is based on our following major findings. (a) EGFR is constitutively activated in Cr(VI)-transformed BEAS-2B cells and in lung tissues from animals and a cancer patient exposed to Cr(VI). (b) EGFR activation initiates autophagy and causes autophagy deficiency by blocking the fusion between autophagosomes and lysosomes, leading to p62 accumulation and constitutive Nrf2 activation in Cr(VI)-transformed cells. (c) Piperlongumine, a natural compound, activates inducible Nrf2 in normal cells and inhibits constitutive activations of EGFR and Nrf2 in Cr(VI)-transformed cells. The central hypothesis is that piperlongumine activates inducible Nrf2, leading to decreased level of ROS and inhibition of malignant transformation of normal cells and that this compound inhibits EGFR activation, resulting in blockages of autophagy process, constitutive activations of p62 and Nrf2, and tumorigenesis of Cr(VI)-transformed cells. Aim 1 will test the hypothesis that EGFR activation initiates autophagy by activating TFEB, which up-regulates Beclin 1 and p62 and generates autophagosomes. Aim 2 will test the hypothesis that EGFR down-regulates LAMP2a, resulting in inhibition of the fusion between autophagosomes and lysosomes, leading to accumulation of p62 and constitutive Nrf2 activation at cellular, animal, and human levels. Aim 3 will demonstrate (a) piperlongumine activates inducible Nrf2, decreases ROS, and inhibits Cr(VI)-induced malignant transformation of normal cells; (b) this natural compound inhibits EGFR activation, autophagy initiation and autophagy deficiency, constitutive activations of p62 and Nrf2, and tumorigenesis of Cr(VI)-transformed cells; and (c) piperlongumine protects from tumor formation in animals exposed to Cr(VI).